JIA-2019-11

2437 Chalisa Chaengsakul et al. Journal of Integrative Agriculture 2019, 18(11): 2435–2445 (15±1)°C in (50±5)% RH until they were used in this experiment. Before the experiment began, the seeds were graded to select only seeds that weighed between 250 and 350 mg and were stored at (15±1)°C in darkness prior to initiation of the experiment. 2.2. Storage conditions and treatments Natural ageing during storage in the laboratory was investigated. Subsamples were placed in polyethylene plastic (PE) bags and stored on a laboratory bench under ambient conditions (~27°C and 50–80%RH) and monitored using a USB data logger (Centor Thai, Bangkok, Thailand). For controlled conditions, seeds were stored in SGB Premium-25RZ GrainPro ® SuperGrainbag ® (GrainPro, Zambales, the Philippines) at (15±1)°C and (50±5)% RH. The seed was stored for 12 months and samples were taken at 3-month intervals for seed quality testing. The effects of artificial ageing, a hot-humid treatment, were tested based on seed quality, ethanol production, and mitochondrial-related gene expression. Seed samples were subjected to ageing in a chamber for different periods (12, 24, and 48 h) in 500 mL glass bottles with 70 mL water to maintain 100% RH, at 42°C. The effect of seed borne-pathogens on ethanol production in fast ethanol assay was also investigated. Sub-samples were surface-sterilised in 1.0% sodium hypochlorite for 10 min and subsequently washed with sterile water for 10 min. Sterilised seeds were air-dried on a thin layer of filter paper for approximately 5 h; thereafter, the seed moisture content (SMC) was reduced to 12% using zeolite seed drying beads ® (Centor Thai, Bangkok, Thailand). Unsterilised seeds were used as a control. Seed quality tests, fast ethanol assay and qPCR were investigated after a disinfection process. 2.3. Seed quality tests Agermination test, accelerated ageing (AA) test, and radicle emergence (RE) test were used for seed quality evaluation with all the samples in this experiment. Germination tests were performed using papers (BP) and techniques (ISTA 2015) with four replicates of 50 seeds. The seeds in rolled paper towels were kept in polyethylene plastic bags and closed boxes and then placed in a cabinet germinator (Seedburo Equipment, IL, USA). The RH in the germinator was maintained very near saturation and the temperature was set to 25°C. Seeds were scored as germinated when normal seedlings were visible according to ISTA (2006). A normal seedling is seedlings with all their essential structures well developed, complete, in proportion and healthy or they show an otherwise satisfactory and balanced development comparable to that of intact seedlings of the same test in case of seedlings showing certain slight defects of their essential structures. Abnormal seedlings would not show the potential to develop into a normal plant when grown in this favourable conditions. Normal seedlings were counted at 4 and 7 days after setting to germinate. The AA test is used as seed vigour test. In this experiment, the AA test followed ISTA (1995). In brief, one layer deep of seed sample was placed on the grille in AA boxes within the ageing chamber at 42°C. The 72-h ageing period started with the placement of the seeds into the ageing chamber. After ageing, four replicates of 50 seeds were used to set up the germination test. The germination tests of each sample were done within 1 h after removal from the ageing chamber. Testing conditions for the normal seedling test were those outlined in the germination test. For the RE test, the vigour level of maize seed was determined by early counts of radicle emergence (~2 mm in length) using the RE test described by ISTA (2015). However, the only difference was the use of four replicates of 50 seeds and incubation at (25±2)°C. In this experiment, radicle emergences was counted at 50 h after setting to germinate. 2.4. Fast ethanol assay The headspace ethanol of a moist seed sample produced during heated incubation, as an indicator of inefficient mitochondria, was determined using a fast ethanol assay. Four replicates of each sample were used to measure ethanol production using the assay that was modified from Kodde et al . (2011). In brief, 3 g of seed sample was placed in 20 mL GC vials. Then, deionized water was added to achieve 20% SMC. The vials were sealed with aluminium crimp caps with 3-mm thick butyl rubber and Teflon septa immediately after adding water. The GC vials containing wetted seeds were incubated at 70°C. The ethanol concentration in the headspace was measured at 1.5 h after incubation using a modified breath analyser (Alcotest 6810 agri; Dräger Safety AG & Co. KGaA; Lübeck, Germany). The needle was shortened to approximately 2.5 cm to allow for pressure equilibration in the vial during its insertion. The device collected a subsample of approximately 0.3 mL from the headspace for every measurement. The modified breath analyser was calibrated semi-annually by the supplier. 2.5. Isolation of total RNA, cDNA synthesis, and qPCR analysis Total RNA isolation from the maize seed was extracted using